Answer the following questions
Botany : Principles and Processes of Biotechnology
17. How do you use the biotechnology in modern practice?
Answer: Modern biotechnology embraces all methods of genetic
modification by recombinant DNA and cell fusion technology. The major focus of
biotechnology are:
(i) Fermentation for production of acids, enzymes, alcohols, antibiotics,
fine chemicals, vitamins and toxins.
(ii) Biomass for bulk production of single cell protein , alcohol, and
biofuel.
(iii) Enzymes as
biosensors, in processing industry.
(iv) Biofuels for
production of hydrogen, alcohol, methane.
(v) Microbial inoculants as biofertiliser, and nitrogen fixers
(vi) Plant and animal
cell culture for production of secondary metabolites, monoclonal
antibodies.
(vii) Recombinant DNA
technology for production of fine chemicals, enzymes, vaccines, growth
hormones, antibiotics, and interferon.
(viii) Process engineering
- tools of biotechnology is used for effluent treatment, water recycling.
18. What are the materials used to grow microorganism like Spirulina?
Answer: (i) Spirulina can be
grown easily on materials like waste water from potato processing plants
(containing starch), straw, molasses, animal manure and even sewage, to produce
large quantities and can serve as food rich in protein, minerals, fats,
carbohydrate and vitamins.
(ii) Such utilization also reduces environmental pollution.
(iii) 250 g of Methylophilus
methylotrophus, as its high rate of biomass production and growth, can be
expected to produce 25 tonnes of protein.
19. You are working in a biotechnology lab with a becterium namely E.coli. How will you cut the nucleotide sequence? explain it.
Answer: (i) A restriction enzyme or restriction endonuclease is an
enzyme that cleaves DNA into fragments at or near specific recognition sites
within the molecule known as restriction sites.
(ii) Based on their mode of action restriction enzymes are
classified into Exonucleases and Endonucleases.
(a) Exonucleases are enzymes which removenucleotides one at a
time from the end of a DNA molecule.
(b) Endonucleases are enzymes which break the internal
phosphodiester bonds within a DNA molecule.
There are three main classes of restriction endonuclease:
Type I, Type II and
Type III
They differ slightly
by their mode of action.
Based on the position
at which we wish to cut the nucleotide sequence and type of cleavage the
corresponding restriction endonuclease may be used.
20. What are the enzymes you can used to cut terminal end and internal phospho di ester bond of nucleotide sequence?
Answer: A restriction enzyme or restriction endonuclease is an
enzyme that cleaves DNA into fragments at or near specific recognition sites
within the molecule known as restriction sites.
(i) Exonucleases are enzymes which remove nucleotides one at a
time from the end of a DNA molecule.
Example: Bal 31, Exonuclease III.
(ii) Endonucleases are enzymes which break the internal
phosphodiester bonds within a DNA molecule.
Example: Hind II, EcoRI, TaqI.
21. Name the chemicals used in gene transfer.
Answer: Chemical mediated gene transfer: Certain chemicals like polyethylene glycol
(PEG) and dextran sulphate induce DNA uptake into plant protoplasts.
22. What do you know about the word pBR332?
Answer: pBR 322 Plasmid:
(i) pBR 322 plasmid is a reconstructed plasmid and most widely
used as cloning vector; it contains 4361 base pairs.
(ii) In pBR, p denotes
plasmid, B and R respectively the names of scientist B Oliver and Rodriguez who developed this plasmid.
The number 322 is the number of plasmid developed from their laboratory.
(iii) It contains ampR and tetR two
different antibiotic resistance genes and recognition sites for several
restriction enzymes. (Hind III, EcoRI,
BamH I, Sal I, Pvu II, Pst I, Cla I), ori and antibiotic resistance genes.
ampR -
Ampicillin Resistance Gene
tetR -
Tetracycline Resistance Gene
pBR 322
(iv) Rop codes for the proteins involved in the replication of
the plasmid.
23. Mention the application of Biotechnology.
Answer: (i) Biotechnology is one of the most important applied interdisciplinary sciences of
the 21st century. It is
the trusted area that enables us to find the beneficial way of life.
(ii) Biotechnology has wide applications in various sectors like
agriculture, medicine, environment and commercial industries.
(iii) This science has an invaluable outcome like transgenic varieties of plants e.g.
transgenic cotton (Bt-cotton), rice, tomato, tobacco, cauliflower, potato and
banana.
(iv) The development of transgenics as pesticide resistant,
stress resistant and disease resistant varieties of agricultural crops is the
immense outcome of biotechnology.
(v) The synthesis of human
insulin and blood protein in E.coli and utilized for insulin deficiency
disorder in human is a breakthrough in biotech industries in medicine.
(vi) The synthesis of
vaccines, enzymes, antibiotics, dairy products and beverages are the products
of biotech industries.
(vii) Biochip based biological computer is one of the successes of bio
technology.
(viii) Genetic engineering involves genetic manipulation, tissue
culture involves aseptic cultivation of totipotent plant cell into plant clones
under controlled atmospheric conditions.
(ix) Single cell protein
from Spirulina is utilized in food
industries.
(x) Production of secondary
metabolites, biofertilizers, biopesticides and enzymes.
(xi) Biomass energy, biofuel, bioremediation, phytoremediation
for environmental biotechnology.
24. What are restriction enzyme. Mention their type with role in Biotechnology.
Answer: Restriction Enzymes:
(i) A restriction
enzyme or restriction endonuclease is
an enzyme that cleaves DNA into fragments at or near specific recognition sites
within the molecule known as restriction sites.
(ii) Based on their mode of action restriction enzymes are classified into Exonucleases and Endonucleases.
(a) Exonucleases are enzymes which remove nucleotides one at a
time from the end of a DNA molecule.
Example: Bal 31, Exonuclease III.
(b) Endonucleases are enzymes which break the internal
phosphodiester bonds within a DNA molecule.
Example: Hind II, EcoRI, TaqI.
Restriction endonuclease: Molecular scissors:
(i) The restriction enzymes are called as molecular scissors.
These act as foundation of recombinant DNA technology.
(ii) There are three
main classes of restriction endonuclease : Type I, Type II and Type III,
which differ slightly by their mode of action.
(iii) Only type II enzyme is preferred for use in recombinant
DNA technology as they recognise and cut DNA within a specific sequence
typically consisting of 4-8 bp. Example: EcoRI.
(iv) The restriction enzyme Hind
II always cut DNA molecules at a point of recognising a specific sequence
of six base pairs. This sequence is known s recognition sequence. Today more
than 900 restriction enzymes that have been isolated from over 230 strains of
bacteria with different recognition sequences.
(v) Restriction endonucleases are named by a standard
procedure.
(vi) For example, EcoRI
is from Escherichia (E) coli (co), strain RY 13 (R) and first endonuclease (I)
to be discovered.
(vii) It contains 2 different antibiotic resistance genes and
recognition site for several restriction enzymes. This sequence is referred to
as a restriction site and is generally -palindromic which means that the
sequence in both DNA strands at this site read same in 5’ - 3’ direction and in
the 3’-5’ direction.
(viii) The exact kind of cleavage produced by a restriction
enzyme is important in the design of a gene cloning experiment.
(ix) Some cleave both strands of DNA through the centre
resulting in blunt or flush end. These are known as symmetric
cuts.
(x) Some enzymes cut in a way producing protruding and recessed
ends known as sticky or cohesive end. Such cut are called
staggered or asymmetric cuts.
Sticky and Blunt ends
25. Is their any possibilities to transfer a suitable desirable gene to host plant without vector? Justify your answer.
Answer: Direct or Vectorless Gene Transfer:
In the direct gene
transfer methods, the foreign gene of interest is delivered into the host plant
without the help of a vector. The following are some of the common methods of
direct gene transfer in plants.
a. Chemical mediated gene transfer: Certain chemicals
like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into
plant protoplasts.
b. Microinjection: The DNA is directly injected into the nucleus using fine
tipped glass needle or micro pipette to transform plant cells. The protoplasts
are immobilised on a solid support (agarose on a microscopic slide) or held
with a holding pipette under suction.
c. Electroporation: Methods of Gene
Transfer: A pulse of high voltage is applied to protoplasts, cells or
tissues which makes transient pores in the plasma membrane through which uptake
of foreign DNA occurs.
d. Liposome mediated method of gene transfer: Liposomes the
artificial phospholipid vesicles are
useful in gene transfer. The gene or DNA is transferred from liposome into
vacuole of plant cells. It is carried out by encapsulated DNA into the vacuole.
This technique is advantageous because the liposome protects the introduced DNA
from being damaged by the acidic pH and protease enzymes present in the
vacuole. Liposome and tonoplast of vacuole fusion resulted in gene transfer.
This process is called lipofection.
e. Biolistics: The foreign DNA is coated onto the surface of minute gold
or tungsten particles (1-3 µm) and bombarded onto the target tissue or cells
using a particle gun (also called as gene gun/micro projectile gun/shotgun).
Then the bombarded cells or tissues are cultured on selected medium to
regenerate plants from the transformed cells.
26. How will you identify a vectors?
Answer: (i) Major component of a gene cloning experiment is a vector
such as a plasmid.
(ii) A Vector is a small DNA molecule capable of
self-replication and is used as a carrier and transporter of DNA fragment which
is inserted into it for cloning experiments.
(iii) Vector is also called cloning vehicle or cloning DNA.
Vectors are of two types:
(a) Cloning Vector, and
(b) Expression Vector.
Cloning vector is used
for the cloning of DNA insert inside the suitable host cell. Expression vector
is used to express the DNA insert for producing specific protein inside the
host.
Properties of Vectors:
Vectors are able to
replicate autonomously to produce multiple copies of them along with their DNA
insert in the host cell.
(i) It should be small in size and of low molecular weight,
less than 10 Kb (kilo base pair) in size so that entry/transfer into host cell
is easy.
(ii) Vector must contain an origin of replication so that it can
independetly replicate within the host.
(iii) It should contain a suitable marker such as antibiotic
resistance, to permit its detection in transformed host cell.
(iv) Vector should have unique target sites for integration with
DNA insert and should have the ability to integrate with DNA insert it carries
into the genome of the hostcell. Most of the commonly used cloning vectors have
more than one restriction site. These are Multiple Cloning Site (MCS) or
polylinker. Presence of MCS facilitates the use of restriction enzyme of
choice.
The following are the
features that are required to facilitate cloning into a vector.
1. Origin of replication (ori): This is a sequence
from where replication starts and piece of DNA when linked to this sequence can
be made to replicate within the host cells.
2. Selectable marker: In addition to ori the vector requires a
selectable marker, which helps in identifying and eliminating non transformants
and selectively permitting the growth of the transformants.
3. Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single, recognition sites for the commonly used restriction enzymes.
27. Compare the various types of Blotting techniques.
Answer: Southern Blotting :
The transfer of DNA from agarose gels to nitrocellulose membrane.
Northern Blotting: The transfer of RNA to nitrocellulose membrane.
Western Blotting : Electrophoretic transfer of Proteins to nitrocellulose membrane.
28. Write the advantages of herbicide tolerant crops.
Answer: Advantages of Herbicide Tolerant Crops:
(i) Weed control improves higher crop yields.
(ii) Reduces spray of herbicide.
(iii) Reduces competition between crop plant and weed.
(a) Use of low toxicity
compounds which do not remain active in the soil.
(iv) The ability to conserve soil structure and microbes.
29. Write the advantages and disadvantages of Bt cotton.
Answer: Advantages of Bt cotton:
(i) Yield of cotton is increased due to effective control of
bollworms.
(ii) Reduction in insecticide use in the cultivation of Bt
cotton.
(iii) Potential reduction in the cost of cultivation.
Disadvantages of Bt cotton:
(i) Cost of Bt cotton seed is high.
(ii) Effectiveness up to 120 days after that efficiency is
reduced.
(iii) Ineffective against sucking pests like jassids,aphids and
whitefly.
(iv) Affects pollinating
insects and thus yield.
30. What is bioremediation? give some examples of bioremediation.
Answer: Bioremediation:
It is defined as the
use of microorganisms or plants to clean up environmental pollution. It is an
approach used to treat wastes including wastewater, industrial waste and solid
waste. Bioremediation process is applied to the removal of oil, petrochemical
residues, pesticides or heavy metals from soil or ground water. In many cases,
bioremediation is less expensive and more sustainable than other physical and
chemical methods of remediation. Bioremediation process is a cheaper and
eco-friendly approach and can deal with lower concentrations of contaminants
more effectively. The strategies for bioremediation in soil and water can be as
follows:
(i) Use of indigenous microbial population as indicator
species for bioremediation process.
(ii) Bioremediation with the addition of adapted or designed
microbial inoculants.
(iii) Use of plants for bioremediation - green technology.
Some examples of bioremediation technologies are:
(i) Phytoremediation - use of plants to bring about remediation of
environmental pollutants.
(ii) Mycoremediation - use of fungi to bring about remediation of environmental
pollutants.
(iii) Bioventing is
the process that increases the oxygen or air flow to accelerate the degradation
of environmental pollutants.
(iv) Bioleaching is
the use of microorganisms in solution to recover metal pollutants from
contaminated sites.
31. Write the benefits and risk of Genetically Modified Foods.
Answer: GM Food - Benefits:
(i) High yield without pest.
(ii) 70% reduction of pesticide usage.
(iii) Reduce soil pollution problem.
(iv) Conserve microbial population in soil.
Risks - believed to
(i) Affect liver, kidney function and cancer.
(ii) Hormonal imbalance and physical disorder.
(iii) Anaphylactic shock (sudden hypersensitive reaction) and
allergies.
(iv) Adverse effect in immune system because of bacterial
protein.
(v) Loss of viability of seeds show in terminatorseed
technology of GM crops.
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